With the coming of age of recombinant DNA technology attention has increasingly been focused on the synthesis of oligonucleotides for various purposes, e.g., as hybridization probes for use in locating complementary DNA made by reverse transcription from purified messenger RNA, as primers in the controlled conversion of single to double-stranded DNA, as plasmidic control regions useful in the bacterial expression of useful proteins, as "linkers" for interpolating heterologous DNA into plasmids, as genes encoding useful proteins that themselves may be bacterially expressed, and so on. In each such case DNA fragments have hitherto been assembled by condensation of nucleotides or oligonucleotides according to a sequential plan dictated by the nucleotide sequence of the desired end product. As one example, the known amino acid sequence of the useful compound somatostatin has permitted design and synthesis of a corresponding gene, which could then be inserted in a bacterial plasmid so as to permit bacterial production of the protein encoded. K. Itakura, et al., Science 198, 1056-1063 (1977).
The construction of oligonucleotides entails phosphorylation of a nucleosidic moiety to form the corresponding 3'-phosphate, which is then condensed with a further, suitably protected nucleosidic moiety to yield a di- or polynucleotide in which the original nucleosidic moieties are linked by a phosphodiester bridge. In the so-called "triester" method the third functionality of the phosphate is protected prior to the condensation reaction to prevent undue side reactions and to neutralize charge so as to permit silica gel chromatography techniques in product purification and recovery. See, e.g., K. Itakura et al., Can. J. Chem. 51, 3649-3651 (1973) and the somatostatin work previously referred to. A typical series of steps in oligonucleotide constructions typical of past triester practice may be represented as follows, "B" being the characteristic base moiety of the nucleoside involved, X a protecting group for the 5'-OH, and R, e.g., p-chlorophenoxy: ##STR2## In the foregoing scheme the possibility exists in the first reaction of byproduct formation owing to e.g., multiple phosphorylation. More to the point, the intermediate product 3 is charged, so that the .beta.-cyanoethanol reactant in the following step must be used in considerable (e.g., 5X) excess if the presence of a polar nucleotidic moiety is to be avoided when product 4 comes to be purified and, of course, workup and purification of 4 is in any event complicated by the excess remaining. Finally, end product 5 is itself charged, and hence cannot be purified in silica gel chromatography, an otherwise highly convenient tool.
A need has accordingly existed for improved means of DNA and other oligonucleotide synthesis, so as to diminish recovery losses, otherwise enhance yields, and to permit more rapid synthesis of key materials in health-related and other fields.